The chemokine receptor CXCR4 and its ligand CXCL12 play a critical role in breast cancer metastasis. Our preliminary work indicates that CXCR4 mediates novel signaling pathways that regulate the CXCL12-induced chemotaxis, chemoinvasion, and adhesion of breast cancer cells. Given the importance of metastasis as the major cause of increased morbidity and eventual mortality in breast cancer patients, understanding of how signaling molecules control the events that lead to metastasis is of fundamental importance. We have shown that a key regulatory signaling molecule linked to CXCR4-mediated breast cancer cell chemotaxis and chemoinvasion is Cbl. The first specific aim of this proposal is to characterize the interaction between Cbl and the CXCR4 receptor. We will also characterize the role of Cbl in modulating CXCR4-mediated chemotactic signaling pathways. Moreover, we are developing innovative strategies to block the CXCR4-mediated metastasis of breast cancer cells. In this regard, we have shown that a novel biological molecule that binds to the Robo receptor, called Slit, blocks CXCL12-induced chemotaxis, chemoinvasion and adhesion, the fundamental components that promote the metastasis of breast cancer cells. The main objective of this project is to analyze the role of Slit as an anti-metastatic factor in breast cancer cells. We hypothesize that: 1) Slit treatment will inhibit breast cancer cell metastasis to the lungs and lymph nodes; 2) Slit induces cross-talk between Robo and the CXCR4 receptor; and 3) Slit inhibits metalloproteinase secretion by blocking the functions of RAFTK/Pyk2 and beta-catenin. To test these hypotheses, we will analyze the regulatory region within the Slit and Robo molecules that mediate anti-metastatic functions. In Aim 2, we will study the effects of different truncated Slit molecules in the chemotaxis, chemoinvasion, and adhesive properties of various breast cancer cells. After in vitro characterization, we will study the in vivo effects of Slit and its truncated form on the pathogenesis of breast cancer metastasis in an in vivo model system. In Aim 3, we will define the domain on the cytoplasmic tail of this Robo receptor that interacts with the CXCR4 receptor. We will also study the effect of the truncated form of the Robo receptor on breast cancer metastasis. In this aim, we will also define the role of RAFTK/Pyk2 and beta-catenin on the Slit-mediated downregulation of matrix-metalloproteinase 2 and 9 secretion and other anti-chemotactic and chemoinvasive mechanisms. These studies will help us to identify CXCL12-induced and Slit-mediated chemotactic/chemoinvasive pathways that may become novel targets for the treatment of breast cancer metastasis. Furthermore, innovative therapeutic strategies to prevent breast cancer metastasis can be anticipated based on these studies of Slit/Robo function.